The new inhibitors were synthesized and tested using high throughput enzymatic and antiviral assays. These collaborative efforts have led to the discovery of highly potent protease inhibitors that retain potency against clinically relevant multidrug-resistant protease variants. The Monogram Biosciences’ PhenoSenseTM assays demonstrated that these inhibitors were highly effective against a diverse panel of wild-type and drug-resistant viruses. Further development of these protease inhibitors may lead to more effective treatments against drug-resistant HIV-1.

In a recent study, we designed highly potent HIV-1 protease inhibitors (PIs) using the substrate envelope model, which confines inhibitors within the consensus volume of natural substrates, providing inhibitors less susceptible to resistance because a mutation affecting such inhibitors will simultaneously affect viral substrate processing. The designed PIs share a common chemical scaffold but utilize various moieties that optimally fill the substrate envelope, as confirmed by crystal structures. The designed PIs retain robust binding to MDR protease variants and display exceptional antiviral potencies against different clades of HIV as well as a panel of 12 drug-resistant viral strains. The substrate envelope model proves to be a powerful strategy to develop potent and robust inhibitors that avoid drug resistance (Nalam et al., 2013).

Binding Affinities of APV, DRV, and the Ten Designed Protease Inhibitors to WT and Drug-Resistant Variants of HIV-1 Protease, determined by a FRET-based enzymatic assay; Ki values are the average of at least three independent measurements.

Resistance Profiles of DRV and the Ten PIs that were DesignedAntiviral potencies (EC50 values) were obtained for WT HIV from clades A, B, and C, and 12 different patient-derived drug-resistant variants of the virus.

Synthesis and SAR Studies of HCV NS3/4A Protease Inhibitors

We have applied the substrate envelope approach to decipher drug resistance against HCV NS3/4A protease inhibitors. We have discovered that, similar to HIV protease inhibitors, drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding. These findings have led to the development of a substrate envelope model for HCV NS3/4A protease, which we have used to understand the molecular basis of drug resistance against NS3/4A protease inhibitors. We are currently engaged in exploiting the HCV NS3/4A substrate envelope model to rationally design more robust protease inhibitors that are less susceptible to resistance.

The available antiviral therapies, which include PEGylated interferon, ribavirin, and one of the HCV NS3/4A protease inhibitors telaprevir or boceprevir, are ineffective for some patients and cause severe side effects. More potent NS3/4A protease inhibitors are in clinical development, but the long-term effectiveness of these drugs is challenged by the development of drug resistance. Recently, we investigated the role of macrocycles in the susceptibility of NS3/4A protease inhibitors to drug resistance in asunaprevir, danoprevir, vaniprevir, and MK-5172, with similar core structures but varied P2 moieties and macrocyclizations. Linear and macrocyclic analogues of these drugs were designed, synthesized, and tested against wild-type and drug-resistant variants R155K, V36M/R155K, A156T, and D168A in enzymatic and antiviral assays. Macrocyclic inhibitors were generally more potent, but the location of the macrocycle was critical for retaining activity against drug-resistant variants: the P1–P3 macrocyclic inhibitors were less susceptible to drug resistance than the linear and P2–P4 macrocyclic analogues. In addition, the heterocyclic moiety at P2 largely determined the inhibitor resistance profile, susceptibility to drug resistance, and the extent of modulation by the helicase domain. Our findings suggest that to design robust inhibitors that retain potency to drug-resistant NS3/4A protease variants, inhibitors should combine P1–P3 macrocycles with flexible P2 moieties that optimally contact with the invariable catalytic triad of this enzyme (Ali et al., 2013).